1. Field of Invention
The present invention relates to genes and proteins that can be used in vaccination, detection and identification of spotted fever rickettsiae infection. More particularly, the invention relates to specific nucleotide sequences encoding a highly specific and immunogenic portion of the outer membrane protein A (OmpA) of Rickettsia rickettsii. The polypeptide sequence can be utilized in diagnostic and detection assays for spotted fever rickettsiae and as an immunogen component in vaccine formulations against spotted fever Rickettsia. 
2. Description of the Prior Art
Rickettsiae are Gram-negative, obligate intracellular parasites of the genus Rickettsia. They are responsible for many spotted fever group Rickettsia infection, and typhus group infection [1]. Of the spotted fever group, Rocky Mountain spotted fever is the most severe and most frequently reported rickettsial illness in the United States. It also occurs in Mexico and in central and south America. The disease is caused by Rickettsia rickettsii, a species of bacteria that is transmitted to humans by ixodid (hard) ticks. Rickettsiae target vascular endothelium. Other named species of the spotted fever group and their geographic distributions are listed in table 1.
Table 1 of Human Disease around the world caused by spotted fever group Rickettsiae. (http://www.cdc.gov/ncidod/dvrd/rmsf/Epidemiology.html, Apr. 1, 2008).
OrganismDisease or PresentationGeographic LocationRickettsia rickettsiiRocky Mountain spotted feverNorth, Central and SouthAmericaRickettsia conoriiMediterranean spotted fever,Europe, Asia, Africa, India,boutonneuse fever, Israeli spotted fever,Israel, Sicily, RussiaAstrakhan fever, Indian tick typhusRickettsia akariRickettsialpoxWorldwideRickettsia sibiricaSiberian tick typhus, North Asian tickSiberia, People's Republic oftyphusChina, Mongolia, EuropeRickettsia australisQueensland tick typhusAustraliaRickettsia honeiFlinders Island spotted fever, Thai tickAustralia, South Eastern AsiatyphusRickettsia africaeAfrican tick-bite feverSub Saharan Africa,CaribbeanRickettsia japonicaJapanese or Oriental spotted feverJapanRickettsia felisCat flea rickettsiosis, flea borne typhusWorldwideRickettsia slovacaNecrosis, erythema, lymphoadenopathyEuropeRickettsiaMild spotted feverChina, Asian Region ofheilongjaiangensisRussiaRickettsia parkeriMild spotted feverUSA
Rocky Mountain spotted fever (RMSF) can be treated easily with antibiotics. If the patient is treated within the first 4-5 days of the disease, fever generally subsides within 24-72 hours. However, it is very difficult to make a clinical diagnosis in the disease's early stages. The early clinical presentation of Rocky Mountain spotted fever is nonspecific and may resemble a variety of other diseases including influenza, measles, and rubella, as well as other rickettsial diseases [2]. The classic symptoms of RMSF are sudden onset of fever, headache, and muscle pain, followed by development of rash, abdominal pain, joint pain, and diarrhea [2]. However, this combination of symptoms is not always detected when the patient initially presents for care. Without prompt and appropriate treatment, the disease can be fatal. Long-term health problems following acute Rocky Mountain spotted fever infection include partial paralysis of the lower extremities, gangrene requiring amputation of fingers, toes, or arms or legs, hearing loss, loss of bowel or bladder control, movement disorders, and language disorders. These complications are most frequent in persons recovering from severe, life-threatening form of the disease, often following lengthy hospitalizations. Therefore, rapid and accurate diagnosis and treatment at the onset of the disease is desirable.
Serologic assays are the most widely available and frequently used methods for confirming cases of Rocky Mountain spotted fever. The indirect immunofluoresence assay (IFA) is generally considered the reference standard in Rocky Mountain spotted fever serology and is the test currently used by Center of Disease Control and most state public health laboratories, but other well validated assays can also be used in diagnosis, including indirect hemagglutination assay (IHA), ELISA, latex agglutination, and dot immunoassays [2].
IFA can be used to detect either IgG or IgM antibodies. Blood samples taken at early (acute) and late (convalescent) stages of the disease are the preferred specimens for evaluation. Most patients demonstrate increased IgM titers by the end of the first week of illness. Diagnostic levels of IgG antibody generally do not appear until 7-10 days after the onset of illness. The value of testing two sequential serum or plasma samples together to show a rising antibody level is very important in confirming acute infection with rickettsial agents because antibody titers may persist in some individuals for years after the original exposure to cross-reactive rickettsial agents. IgG antibodies are more specific and reliable since other bacterial infections can also cause elevations in riskettsial IgM antibody titers. One of the disadvantage in using IFA to diagnose Rocky Mountain spotted fever is that most patients visit their physician relatively early in the course of the illness, before the presences of diagnostic antibody level. In addition, variations in the endpoint titer may occur due to differences in the quality of the microscope, the quality of the anti-immunoglobulin conjugate, and the experience of the technician.
Both IHA and latex agglutination rely on a common source of rickettsial antigen, extracted from R. rickettsii. This antigenic material is coated on sheep or human type O erythrocytes for IHA and onto latex beads for latex agglutination. Although IHA test demonstrates the earliest, steepest rise in antibody titer of all serologic tests for RMSF, it is rarely used as the diagnostic method in acute state of the illness. Only 19% of patients with RMSF had an acute titer of 40, which is much lower value than CDC's criterion for using a single titer indicating a probable diagnosis (≧128) [2]. The latex agglutination test is technically simple and rapid and requires no elaborate equipment. However, latex agglutination test is inappropriate for serosurveys and is more diagnostically discriminatory for establishing the diagnosis of a recent infection because the detectable antibody has persistent presentation months after the onset of illness [2].
Although routinely used in retrospective confirmatory diagnosis, current serologic methods are not considered appropriate rapid acute diagnostic tests. Very seldom are specific antibodies to R. rickettsii detected during the acute stage of illness when empiric treatment must begin. The most rapid and specific diagnostic assays for Rocky Mountain spotted fever rely on molecular methods like PCR, which can detect DNA of 5-10 rickettsial organisms in a sample. While organisms can be detected in whole blood samples obtained at the acute onset of illness in a few hours, rickettsial DNA is most readily detected in fresh skin biopsies like those used in immunostaining procedures. PCR can also be done on the fixed tissues used in immunostaining, but it is less sensitive than with unfixed tissues. PCR methods can be R. rickettsii-specific but are usually confirmed by DNA sequencing of the amplified gene fragments. Consequently, this procedure is more specific than antibody-based methods which are often only genus or spotted fever group-specific. However, gene amplification requires sophisticated instrumentation and reagents generally not available in most rural medical facilities. In addition, extensive training is required for the end users to achieve accurate and standardized results.
Another approach to Rocky Mountain spotted fever diagnostics is immunostaining. This method is used by taking a skin biopsy of the rash from an infected patient prior to therapy or within the first 48 hours after starting the antibiotic therapy. However, because rickettsiae are focally distributed in lesions of Rocky Mountain spotted fever, this test may not always detect an agent. Even in laboratories with expertise in performing this test, the sensitivity is only about 70% on biopsied tissues because of the scarcity of organisms in some samples.
Two major outer membrane proteins of spotted fever group Rickettsiae, OmpA and OmpB, have been identified as major immunogenic antigens. Outer membrane protein A (OmpA) has an apparent molecular mass of 190 kDa. Immunization with recombinant rOmpA (Rickettsia OmpA) protects guinea pigs against a lethal dose of R. rickettsii. The rOmpA gene of (R. Rickettsia) contains 6747 nucleotides that code for 2249 amino acid protein [5]. Moreover, immunization with recombinant OmpA of R. conorii completely protects guinea pigs against challenge with R. conorii and partially protects against challenge with R. rickettsii [6]. OmpA of R. Rickettsii was also shown to react strongly with sera from patients infected with R. rickettsii and other spotted fever group Rickettsia. Based on these observations, therefore, OmpA is a particularly advantageous target for developing diagnostic reagents against spotted fever group Rickettsia, especially against R. Rickettsii, the causative agent of RMSF.
FIG. 1 shows that fragment X of Omp A is conserved among various spotted fever group Rickettsia. FIG. 2 is a comparison of fragment Y of OmpA from various spotted fever group Rickettsia. 